Medicaments containing gelatin cross-linked with oxidized polysaccharides

ABSTRACT

The present invention relates to a wound dressing comprising a biopolymer matrix comprising gelatin cross-linked with an oxidized polysaccharide. Preferably said oxidized polysaccharide comprises an oxidized dextran or an oxidized xanthan. Preferably said matrix is in the form of a hydrated film, a hydrated or dry foam, dry fibers which may be fabricated into a woven or non-woven tissue, hydrated or dry microbeads, dry powder; or said matrix is covered with a semipermeable film, so as to control the humidity of the wound covered with the dressing, with the permeability chosen so as to maintain this humidity within a therapeutically optimal window. A polysulfated polysaccharide with a M.W. greater than 30,000 kDa is mechanically entrapped during the formation of said matrix.

This application is a 371 of PCT/EP97/002279 filed May 5, 1997.

The present invention relates to a medicament containing a biopolymermatrix comprising gelatin cross linked with oxidized polysaccharides.The material is useful for the covering of a variety of wound types,particularly chronic wounds and burns. The material is also suitable forthe controlled release of drugs. When loaded with suitable growthfactors or wound repair promoting substances, the matrix is useful forthe fabrication of wound dressings for the treatment of a variety ofwound types, particularly chronic wounds and burns.

A very large number of people are suffering from chronic non-healingskin wounds. Worldwide, 8 million people have chronic leg ulcers and 7million people have pressure sores (Clinica 559, 14-17, 1993). In the USalone, the prevalence of skin ulcers is 4.5 million, including 2 millionpressure sore patients, 900,000 venous ulcer patients and 1.6 milliondiabetic ulcer patients (Med Pro Month, June 1992, 91-94). The costinvolved in treating these wounds is staggering and, at an average of$3,000 per patient, reaches over $13 billion per year for the US alone.Burn wounds have a reported incidence of 7.8 million cases per yearworldwide, 0.8 million of which need hospitalisation (Clinica 559). Inthe US there are 2.5 million burn patients per year, 100,000 of whichneed hospitalization and 20,000 of which have burns involving more than20% of the total body surface area (MedPro Month, June 1992).

A common feature in the treatment of these wounds is that they needcovering for optimal healing. The beneficial effect of covering woundsis situated at different levels and is dependent on the type of dressingmaterial used. First, especially with acute wounds, suitable dressingsmay help to achieve haemostasis and thus control blood loss. Secondly,covering effectively shields the wound from the environment, thusprotecting it from microbial contamination. Furthermore, some so-calledocclusive or semi-occlusive wound dressings have the capability ofmaintaining the wound moist, which is beneficial for healing. Finally,some wound dressings may themselves directly promote the healingprocess, for instance because they contain components which directlypromote cell growth or migration or which attract or activate cells fromthe immune system which on their turn secrete growth-promotingsubstances. Other dressings may contain antimicrobial substances, whichare helpful to control infection of the wound.

Over time, a surprisingly wide variety of dressing materials have beenused for wound covering, many of which are currently commerciallyavailable. Each of them has its own indications, dependent on woundtype, depth, size, absence or presence of infection, level of exudateformation, etc.

Cotton gauze, for instance, is widely used as wound dressing. It has theadvantage of being cheap, but the disadvantage of being not occlusiveand sometimes becoming encrusted into the wound. To prevent this, thesedressings are sometimes impregnated with a greasy substance, such asparaffin. A commercially available example of such a dressing isJelonet™ (Smith and Nephew, UK).

Another class of wound dressings are the absorptive hydrogel dressings.These have no occlusive properties, but have a high capacity for theabsorption of exudate and slough. They consist of hydrophilic polymerssuch as gelatin, polysaccharides, polyacrylamide, etc. which swell uponcontact with wound fluid and can absorb several times their own weightof exudate. Commercially available hydrogel dressings include Intrasitegel (Smith and Nephew, UK) and Vigilon (CR Bard, USA). A special type ofhydrogels are the alginates, which are hydrophilic polysaccharidesextracted from seaweed. They are produced as thin non-woven tissues oras ropes. Upon contact with the wound fluid, they turn into a gel whichhas a high absorptive capacity for wound fluid. Examples includeKaltostat (Brit-Cair, UK) and Sorbsan (Steriseal, UK).

Another type of dressings are the occlusive or semi-occlusive dressings.In their simplest form, they usually exist of a thin, flexible plasticmembrane, e.g. from polyurethane. To facilitate application, thesedressings are usually fabricated with a self-adhesive coating. Thesedressings are called occlusive because they limit water evaporation fromthe wound surface, thus keeping it moist. Examples of such dressings areOpsite (Smith and Nephew, UK) and Tegaderm (3M, USA). Examples ofsemi-occlusive dressings are Omiderm (latro Medical Systems, UK) andExkin (Koninklijke Utermohlen, The Netherlands). The latter dressingsallow a slightly higher evaporation rate, resulting in a semi-dry woundsurface.

A more complex type of occlusive dressings are the hydrocolloid (HCD)dressings. These are made up of hydrocolioid particles (e.g. consistingof gelatin, pectin, etc.) embedded in a hydrophobic matrix (e.g. apolyisobutylene). These dressings may be backed with an occlusivemembrane and/or a foam plastic layer. In addition to being occlusive,HCD dressings have a high absorptive capacity, making them very suitablefor the treatment of wounds producing high amounts of exudate. Thesebeneficial properties have made HCD dressings among the mostsuccessfully used dressings for the treatment of chronic ulcerations ofthe skin. Commercially available examples of these dressings includeDuoderm° (Convatec, UK) and Tegasorb™ (3M, USA).

Although highly successful, recent reports suggest that HCD dressingsmay nevertheless induce undesirable side reactions in the treatedtissues. For example, Van Luyn reports that Duoderm E (Convatec, UK),Biofilm (Biotrol SPA, France), Comfeel (Coloplast, Denmark) and Ulcerdressing (Johnson and Johnson, USA), all of which are HCD dressings,fall within the high toxicity class when tested in a methylcelluloseassays using human skin fibroblasts as target cells (Van Luyn, M.Doctoral Thesis, 1992, State University Groningen, The Netherlands; VanLuyn, M., Abstract Book of the joint WHS/ETRS meeting, Amsterdam, 1993p114). All the HCD dressings tested by this author highly inhibited cellgrowth (>70%) and induced strongly deviant morphologies in the survivingcells. Leek et al. (Abstract Book of the Second Annual WHS Meeting,Richmond, Va., USA, p75, 1992) have tested four HCD dressings infull-thickness excisional wounds in pigs. All dressings induceddevelopment of granulomatous lesions between 4 and 10 days post woundingand exhibiting little evidence of resolution at 3 months post wounding.The most severe reaction was obtained with Duoderm and Intrasite HCD.Rosdy and Clauss (J. Biomedical Mat. Res. 24, 363-3777, 1990) found thatthe HCD dressing Granuflex™ (Bristol-Myers Squibb, USA) inducedcytopathic effects on MRC5 fibroblasts and epidermal cells upon directcontact. Young et al. (J. Invest. Dermatol. 97, 586-592, 1991) describein an animal model system the development of deep-seated foreign bodytype reactions and granulomata in healed wounds which were treated withHCD dressings. Our own experiments with the HCD dressing Duoderm™ showthat this dressing results in a marked and chronic inflammatory responsewhen placed in full thickness wounds in pigs.

The above mentioned data suggest that, while HCD dressings may promotewound healing in the short term, their use is often associated withundesirable inflammatory effects. Therefore, it is clear that there is aneed for a wound dressing displaying the beneficial properties of HCDdressings, yet resulting in substantially less chronic inflammation orforeign body response. Such a wound dressing would stimulate granulationtissue formation, be absorptive and preferably be biodegradable within alimited time frame.

Gelatin, which is a denatured form of the protein collagen, has beenused in a variety of wound dressings. Since gelatin gels have arelatively low melting point, they are not very stable at bodytemperature. Therefore, it is imperative to stabilize these gels byestablishing cross-links between the protein chains. In practice, thisis usually obtained by treating the gelatin with glutaraldehyde orformaldehyde. Thus cross-linked gelatin may be fabricated into drysponges which are useful for inducing haemostasis in bleeding wounds.Commercially available examples of such sponges include Spongostan°(Ferrosan, Denmark) and Gelfoam (Upjohn, USA). A major disadvantage ofthese sponges is that the cross-linking agent used (formaldehyde orglutaraldehyde) is toxic for cells. The negative effect ofglutaraldehyde cross-linking is exemplified, for instance, by thefindings of de Vries et al (Abstract Book of the Second Annual Meetingof the WHS, Richmond, USA, p51, 1992). These authors showed thatglutaraldehyde cross-linked collagen lattices were toxic for cells,whereas the non cross-linked variety was not. Therefore, despite theirbeneficial haemostatic properties, these products are not very optimalas wound dressings for the treatment of problematic wounds such aschronic ulcers or burns. Consequently, a gelatin-based wound dressingwhich uses a different, less toxic, cross-linking technology would bevery desirable. Dextran is a polysaccharides which is also widely usedfor medical purposes, and which may also be used in a wound dressing.For example, PCT publication number WO 94/27647 (Smith and Chakravarty)teaches the fabrication of a polymer composition comprised ofcross-linked dextran, where the cross-linking groups consist of linearimido carbonate or carbonate groups. This polymer can be incorporated ina wound dressing. An important feature of this polymer composition isthat it is hydrolytically labile. This means that hydrated forms of thematerial are inherently unstable, and that the polymer can only bestored for prolonged periods when dehydrated.

Schacht et al., in European patent published under N° 0308330 disclose apolymer composition comprising gelatin, cross-linked with oxidizedpolysaccharides onto which proteins, enzymes or micro-organisms areadditionally immobilized.

Apart from the development of improved dressings, increasing attentionhas been given over the last years to the possible use of growth factorsto promote the healing of wounds, in particular burns and ulcers.Following are but a few of the scientific reports describing the use ofgrowth factors for promoting wound healing in humans. Epidermal GrowthFactor (EGF) has been used for the treatment of skin graft donor sites(Brown et al., N. Engl. J. Med. 321, p76-79, 1989) and chronic ulcers(Brown et al., Plast. Reconstr. Surg. 88, p.189-194, 1991). This samefactor has also successfully been used in ophthalmology for the topicaltreatment of traumatic corneal ulcers (Scardovi et al., Opthalmologica206, p.119-124, 1993) and to promote endothelial wound healing in humancorneas (Hoppenreijs et a., Invest. Ophtalmol. Vis. Sci. 33, p1946-1957,1992). EGF eye drops are commercially available under the trade nameGentel° from Inpharzam S.A. (Cadempino, Switzerland). Basic FibroblastGrowth Factor (bFGF) has been used for the treatment of chronic pressuresores (Robson et al., Ann. Surg. 216, p.401-408, 1992) and for thetreatment of experimentally induced suction blisters in humans (Lyonnetet al., J; Invest. Dermatol. 96, p.1022, 1991). Transforming GrowthFactor β (TGFβ) was found to have beneficial effects in the treatment offull thickness macular holes in human eyes (Glaser et al., Ophthalmology99,n p1162-1173). Platelet Derived Growth Factor (PDGF) was found to bea wound healing stimulator of chronic pressure ulcers in humans (Robsonet al., Lancet 339, p.23-25, 1992). Human Growth Hormone has beenreported to accelerate wound healing in children with large cutaneousburns (Gilpin et al., Ann. Surg. 220, p.19-24, 1994). Platelet lysate,which is a crude preparation containing a mixture of several growthfactors, has also been found to stimulate the healing of chronic ulcers(Knighton et al., Surgery Gyn. Obst. 170, 56-60, 1990). The latterpreparation has been commercialized under the trade name Procuren byCurative Technologies, Inc (USA). Our own studies with crudekeratinocyte lysates, which also contain several cell growth promotingactivities, have shown an increase of the healing speed of burns woundsand an enhancement of epithelialisation of middle ear defects in chronicotorrhea patients and after tympanoplasty.

One common problem with all aforementioned studies is to find anefficient way for the controlled delivery of the active substances tothe wound. In most cases; these substances are simply applied as anaqueous solution, or at best as a formulation in a semi-liquid gel orcream. Using such formulations, most of the active substance is releasedin the wound site very rapidly. Nevertheless, it is known that manygrowth factors are relatively unstable and it is expected that theirhalf life in the wound environment is relatively short. This means thatthere is a need for a device which would allow the controlled release ofthe active substance over a prolonged period, at the same timeprotecting the still unreleased factor from premature degradation. Thiswould significantly lower the cost and increase the efficiency of growthfactor wound therapy by reducing the necessary dose and by securing amore prolonged effect of the active substance, thereby reducing thenumber of applications. Several strategies and materials have beenconsidered for the controlled release of peptide growth factors andsimilar substances. Following are a few of the approaches which havebeen reported in the scientific literature or for which patentapplications have been filed.

One class of controlled release devices consists of syntheticbiodegradable polymers. For instance, poly-lactide-glycolides (PLG) arehydrolytically degradable polymers which can be used for the slowrelease of variable pharmaceutical substances including bioactivemacromolecules such as calcitonin, LHRH, somatostatin, insulin,interferon and vaccines (Lewis, Pharmaceutical manufacturingInternational, 1993, p99-105). Due to the use of organic solvents,incorporation of biologically active peptides or proteins into PLG oftenresults in their inactivation. Although this can be circumvented by theproduction of physical PLG/peptide mixtures (e.g. by compressionmoulding of powder mixes), these may be less suitable as wound dressingsbecause of their rigidity and brittleness.

Apart from synthetic polymers, a wide variety of naturally occurringpolymers, or modifications thereof, have been used for controlledrelease of bioactive peptide factors. An example of this ismethylpyrrolidone chitosan fleeces loaded with bFGF (Berscht et al.,Biomaterials 15, 593-600, 1994). A particular controlled releasecomposition is disclosed in WO 92/09301 by Greisler, which teaches theuse of growth factor-containing fibrin tissue sealant for accelerationof wound healing. Products according to the latter invention wouldprobably be relatively expensive, due to the high cost of commerciallyavailable fibrin glues adhesives.

A frequently used biopolymer for controlled release is also gelatin.Collagen-containing gelatin sponges for protein drug delivery have beendisclosed in patent applications EP 0568334 and WO 93/21908. Golumbek etal., in Cancer Res. 53, p5841-5844 (1993), describe the use of gelatinmicrospheres loaded with IFNγ or GM-CSF as potential cancer therapyvaccines. Cortesi et al. (Int. J. Pharm. 105, p.181-186, 1994) describethe use of gelatin microspheres for the release of syntheticoligonucleotides and PCR-generated DNA fragments. The synthesis ofgelatin microspheres containing Interferon was reported by Tabata andIkada (Pharm. Res. 6, p.422-427, 1989). Shinde and Erhan (Bio-Med. Mat.Eng. 2, p.127-131, 1992) describe flexibilized gelatin films for therelease of insulin.

As discussed above, the commonly used glutaraldehyde or formaldehyde forcross-linking these gelatin-based biomaterials have the disadvantage ofbeing toxic for the cells. In addition to their toxic properties,glutaraldehyde and formaldehyde are also expected to affect thebiological activity of incorporated bioactive protein substances whencross-linking is carried out after addition of these substances to thesystem. Consequently, a gelatin-based slow release device which uses adifferent, less toxic, cross-linking technology would be very desirablefor the fabrication of, for instance, growth factor-medicated wounddressings.

The present invention thus aims at providing a suitable wound dressing.

The present invention also aims at providing a suitable slow orcontrolled release device.

The present invention further aims at methods for producing and usingsaid wound dressings or said controlled or slow release devices.

The present invention relates to the unexpected finding that polymerscomprising gelatin cross-linked with oxidized polysaccharides constituteexcellent medicament such as dressings for the treatment of wounds. Thecross links are formed by Schiff base formation between free aminogroups of the gelatin and aldehyde groups in the polysaccharides. Apolysaccharides particularly suited for use in the present invention isdextran. For the purpose of clarity, oxidized dextran shall be called"dextranox" hereafter, and the polymer composition consisting ofgelatin, cross-linked with oxidized polysaccharides (in particulardextran), shall be called "GDP" (for Gelatin-Dextranox Polymer).

One of the advantages of the presently disclosed medical composition forwound healing purposes is that it is a fully biodegradable material.Nevertheless, since biodegradability is not obtained through the use ofhydrolytically cleavable bonds, the object of our invention issufficiently stable in a hydrated form to allow prolonged storage.Unlike non cross-linked gelatin it also has a melting point sufficientlyhigh to remain on the wound site in an intact form for a sufficientlylong time. Another advantage is that the disclosed wound dressing hassubstantially reduced cytotoxical and inflammatory properties ascompared with existing gelatin-based materials. This is exemplified inexamples 3-5. Yet another advantage is that the material inducesgranulation tissue formation in experimental wounds, a feature which ishighly desirable for the treatment of chronic wounds. A furtheradvantage is that one of the embodiments of the disclosed dressingoffers the possibility to immobilize sulfated dextrans or similarpoly-anionic molecules into the dressing, a modification which enhancesthe binding of incorporated or local heparin binding wound repairmodulating factors.

According to a further aspect, the present invention relates to theunexpected finding that gelatin cross-linked with oxidizedpolysaccharides constitutes an ideal biopolymer matrix for theincorporation and subsequent controlled release of bioactive peptidefactors. Contrary to the anticipations, it was discovered that theincorporated bioactive peptide factors were only marginally,cross-linked to dialdehyde groups of the oxidized polysaccharides.Therefore, pharmaceutically active peptides can be incorporated in thematrix by mixing them with the solubilized gelatin component, followedby addition of the dextranox component to obtain a.stabilizedcross-linked gel containing the peptide in a releasable form. Thisallows a much more elegant, cheaper and controllable production processas compared with the alternative procedure of incorporating the peptidesby a sorption process (e.g. by soaking the dehydrated or partiallydehydrated matrix in a solution containing the peptide). Such medicatedGDP matrices may be used for several therapeutical applications, inparticular for the fabrication of medicated wound dressings, e.g. byloading them with growth factors or other wound repair-enhancingsubstances.

The GDP matrix also functions as an efficient matrix for the controlledrelease of biomolecules, as shown in examples 6-8. Medicated GDPmatrices prepared in this way are useful for a variety of therapeuticalapplications, in particular for the fabrication of medicated wounddressings.

The term "biopolymer matrix" according to the present invention refersto a matrix composed of mixtures of gelatin and oxidized polysaccharidesas defined above having as a basic property that they are biodegradable.

In a preferred embodiment, the proposed wound dressing consists of ahydrated sheet or film of matrix as defined above, backed with anocclusive or semi-occlusive film. Occlusive in this context means thatthe film has a permeability for water which is sufficiently low toprevent desiccation of the wound, yet sufficiently high to preventexcessive accumulation of exudate below the wound dressing.

In another embodiment, the wound dressing is fabricated in the form ofdehydrated microparticles. These microparticles are especially suited tobe applied into deep, highly exudative wounds. By virtue of the highfluid-absorptive capacity of the particles, the wounds may in this waybe cleaned from excess exudate and slough.

In yet another form, the proposed polymer is fabricated into a flexibledehydrated foam. Such a foam may be easily applied onto shallow woundsand also has a high absorptive capacity.

The proposed polymer can also be used for the fabrication of a wounddressing containing one or more wound repair-promoting substances.Examples of such substances are for instance growth factors such as EGF,TGF-α, FGFs, PDGFs, amphiregulin, HB-EGF, betacellulin, TGF-β, IGFs orother mitogens or their antagonists which may modulate the wound repairprocess. Such a medicated wound dressing can be produced in differentforms, including flexible sheets, foams, microparticles, fibers to makeup woven or non-woven tissues, etc. One of the embodiments of theinvention concerns the production of a wound dressing containingmultiple layers, where each layer contains a different active component,so as to achieve a programmed delivery of the different components overtime. In another embodiment, suitable affinity groups are incorporatedinto the polymer matrix, to increase the affinity of the matrix for theincorporated active substances, thus decreasing their release rateand/or to protect them from premature degradation or inactivation.Examples of such affinity groups include heparin or functional analogsof heparin such as dextran sulfate, which have an affinity for heparinbinding growth factors such as the FGFs, amphiregulin and HB-EGF.Possible affinity groups also include monoclonal or polyclonalantibodies or microproteins as obtained through phage display, and whichhave a high and selective affinity for specific growth factors.

The present invention relates more particularly to the finding that GDPconstitutes an excellent material for the preparation of dressingssuitable for the covering and treatment of wounds. In addition, thematerial also displays unexpectedly favourable controlled releaseproperties for the delivery of therapeutic substances, particularly towounds. GDP is prepared by the cross-linking of solubilized gelatin withoxidized dextran. Gelatin is a denatured form of the connective tissueprotein collagen. Several types of gelatin exist, depending on thesource of collagen used, and on the extraction and production processemployed. One type of gelatin is extracted from animal bones, whileanother type is extracted from animal skin. Usually, the animal materialis from bovine or porcine origin. Depending on the extraction process,two types of gelatin can be prepared: the A (or acidic) type, which isprepared by acid hydrolysis of the collagen and which has an isoelectricpoint of about 8, and the B (or basic) type, which is prepared by basichydrolysis of the collagen and which has an isoelectric point of about5. Both types of gelatin are useful for preparation of GDP or matricesas defined above suitable for the present invention. However, becausecross-linking with dextranox is preferably performed at a pH superior tothe isoelectric point of the gelatin, the A type is the most preferredtype of gelatin, because it allows cross-linking at an approximatelyneutral pH of between 6 and 8. An important property of gelatin is thatit forms gels with a certain rigidity. The rigidity of these gels isexpressed by the Bloom number of the gelatin. For the purpose of thisinvention, gelatins with a variety of Bloom numbers are usable. However,Bloom numbers of at least 150, preferably at least 200, more preferablyat least 250 are preferred because they offer GDP of a high mechanicalstrength which can easily be fabricated in films or sheets. Again, sincegelatins of the A type usually have higher Bloom numbers, this type isthe most preferred within the framework of this invention.

The oxidized polysaccharides used in the present invention is preferablyan oxidized dextran. However, it shall be obvious to the person skilledin the art that other polysaccharides with suitable viscosity, molecularmass and oxidation properties can also be used, as described in theabove-mentioned patent application EP 0308330 by Schacht and Nobels, thecontents of which are hereby incorporated by reference. An example ofsuch another polysaccharide is oxidized xanthan. Although differentpolysaccharides are thus conceivable for the purpose of this invention,we shall from hereon only refer to the use of oxidized dextrans (whichwe call dextranox). This is simply for the sake of clarity and should inno way be considered as a limitation with respect to the range ofpossible polysaccharides useful within the framework of the invention.The molecular weight of the dextran used for the fabrication of wounddressings according to the invention is preferably below 5,000,000, morepreferably between 10,000 and 100,000, in such a way that the viscosityof the aqueous solution of the dextran is not too high, for examplebetween 0.1 and 1 Pa.s for a 2% solution (as measured using a BrookfieldLVT viscosimeter operated at 30 cycles).

Oxidation of dextran is a well-known reaction. For instance, oxidationcan be conveniently obtained by treatment with an aqueous solution of asalt of periodic acid, such as sodium periodate. The purpose of theoxidation is to create the formation of reactive dialdehyde residues inthe polysaccharides. Although the oxidation procedure described above ispreferred, it shall be clear to the person skilled in the art that otheroxidation methods leading to the formation of dialdehyde residues arealso possible, for instance, by treatment with periodic acid or leadtetra acetate in an organic solvent such as dimethylsulfoxide. Afteroxidation, the dextranox can be conveniently purified and separated fromlow molecular weight reaction components by classical purificationmethods. Examples to accomplish this include, but are not limited to:precipitation (for instance by addition of acetone, methanol orisopropanol) or dialysis, ultrafiltration or gel permeationchromatography, followed by lyophilisation.

Cross-linking between gelatin and dextranox is accomplished by theformation of so-called Schiff base links between free amino groupspresent on the gelatin (notably on the lysine residues thereof) and thedialdehyde residues on the dextranox. This reaction is performed inaqueous medium and the speed and degree of cross-linking are dependenton a variety of parameters, such as the type of gelatin, theconcentration, degree of dialdehyde substitution and molecular weight ofthe dextranox, the pH, buffer type and the presence of electrolytes inthe reaction medium, etc. Suitable reaction parameters are described forinstance in patent application N° EP 0308330, cited above. For thepurpose of this invention, the percentage of oxidation of the dextranoxis preferably between 5% and 50%. For application of GDP as a slowrelease device for the delivery of proteins, it is preferably between 5%and 20%, more preferably between 5 and 15%, even more preferably between7 and 12%. The concentrations of gelatin and dextranox are preferablybetween about 2% and about 20%, more preferably between 5 and 15%, evenmore preferably between 7 and 12%.

According to the present invention, GDP prepared as described above canbe used for the fabrication of a variety of wound dressings.

According to a preferred embodiment, GDP is fabricated into a thin sheetor film, suitable for application onto a wound surface. There existseveral known technologies to accomplish this. For instance, a solutionof gelatin (kept at a temperature higher then the gelification point ofthe gelatin used, usually >30° C.) can be mixed with a solution ofdextranox and be poured into a suitable cast before any appreciablecross linking takes place. After the cross-linking process is finished,the film can be removed from the cast. Another way to form films is touse one of the processes utilized in the photographic industry for thepreparation of photographic films and papers. For the purpose of thisinvention, the thickness of the films shall preferably range between 0.1and 2 mm, more preferably between 0.3 and 1 mm, although differentlysized films may be appropriate for some applications.

When a film according to the procedure described above is placed onto awound for a prolonged period, it is possible that dehydration stilltakes place because fluid can evaporate from the surface of the film. Toprevent this, the GDP wound dressing film can be additionally covered byone of the commercially available occlusive or semi-occlusive wounddressing films, for example a polyurethane such as Opsite or Tegaderm.However, a better solution is provided according to another preferredembodiment of the present invention where a GDP film is directlylaminated onto a suitable occlusive membrane during the productionprocess. Examples of such membranes are provided in example 2. Forinstance, particularly well suited plastic films are those from thePebax series, such as Pebax 1205, which are produced by Elf. This typeof film has a very low water vapour permeability, making it verysuitable for the fabrication of wound dressings intended for use onrelatively dry wounds. For application on more exudative wounds a higherevaporation rate is desirable, to prevent excessive accumulation offluid under the dressing. In this instance, a backing membrane withhigher water vapour permeability may be preferred, such as thosemanufactured by Utermohlen in The Netherlands (Exkin) or by latroMedical Systems in the UK (Omiderm). To the person skilled in the art itshall be obvious that, depending on the type of wound, the degree ofexudate formation and the desired frequency of dressing change, otherbacking films with different water vapour permeability properties can beused, to obtain an optimal fluid balance at the wound surface.

According to another embodiment, GDP is fabricated into a hydrated ordehydrated particulate wound dressing. Several techniques are known toachieve this. A dry GDP powder or granulate may be produced bydehydration of a solid GDP mass after cross linking, followed bypowdering the dehydrated material. Dehydration may be obtained forinstance by drying in a stream of dry air, lyophilisation, organicsolvent extraction, etc. After the powdering or granulation step,particles of a desired size may be selected, for instance by sievingthrough a series of sieves with a suitable mesh size. For themanufacturing of spherical or substantially spherical GDP particles, onecan generate a spray by pushing a freshly prepared gelatin/dextranoxmixture through an approporiate atomization nozzle. It has to beunderstood that the sizes of the spray drops will vary according to thetype of application and can be determined by choosing the appropriatenozzle type, pressure and capacity for the atomization process. Anotherpossibility is to emulsify a freshly prepared gelatin/dextranox solutionwith a non-water miscible solvent such as an aliphatic or aromatichydrocarbon or an oil, provided this solvent does not contain anyresidues which can react with aldehydes. To create spherical particlesof a larger size, the gelatin/dextranox solution may alternatively beadded dropwise to the non-water miscible solvent. Other techniques toproduce hydrated or dehydrated gel particles, known to the personskilled in the art, may also be used to prepare a particulate wounddressing according to this invention. Such a particulate wound dressingmay be useful for the treatment of a variety of wound types, butespecially for the treatment of relatively deep and highly exudativewounds, such as some chronic ulcers or diabetic foot ulcers or decubituswounds. When applied in a dehydrated form they have the property ofabsorbing exudate. This is a highly desirable feature, since removal ofexcess exudate and slough is an important therapeutical goal withrespect to the prevention of microbial colonization, to the limitationof further necrotization and to the relieve of discomfort for thepatient. Such a particulate wound dressing can also be used in itshydrated form (i.e. by omitting the dehydration process after particlepreparation or by rehydrating dehydrated particles before applicationonto the wound). In this latter form, it can be applied for instance asa paste to wounds which produce less exudate. It shall be obvious that,depending on the needs of a particular wound type, the possibility alsoexists to use the particulate wound dressing in a partially hydratedform. In the latter form, the dressing still would have substantialfluid absorptive properties, yet, by virtue of a certain stickiness, itwould easily be applicable as a paste or be fabricated into a thin film.By adapting the type of gel, wound dressings can be designed that areappropriate for treatment of other wounds such as corneal wounds ordefects, tympanic membrane reconstructions, or other middle earreconstructions, or chronic otorrhea. It shall also be clear that thedehydrated, partially hydrated and fully hydrated forms of theseparticulate wound dressings can be suspended in any suitable aqueous ororganic excipient to facilitate application. Examples of such excipientsinclude, but are not limited to: paraffin oil, vaseline, glycerol, etc.

Another physical form into which GDP wound dressings can be fabricatedis a foam. This can be achieved for instance by adding a suitablebiocompatible detergent to the freshly prepared gelatin/dextranoxmixture, followed by introducing small gas bubbles into the mixture. Thegas can be air, nitrogen, helium or another gas, preferably a gas whichis not water soluble, non-toxic and chemically inert. Other techniquesknown in the art for producing foams are also suitable, provided they donot result in the introduction of non-biocompatible components or do notinterfere with the cross-linking process. Foams can be used either inthe hydrated form, or be also partially or completely dehydrated. Theycan be produced as sheets, rods, plugs, pads, etc., or in any other formwhich is considered suitable for easy application to a wound site.

In a further embodiment, other molecular components may be covalentlyattached to or into the GDP matrix through SIPN technology(semi-interpenetrating polymer network) or a combinatiuon of both.Especially, high molecular weight components can be mechanicallyentrapped within the polymer meshwork, such that through covalentattachment is not always required. These components may consist ofmolecules which have a known affinity for certain growth factors orwound healing-promoting substances. Examples of such components arethose with affinity for heparin binding proteins, such as heparin orfunctional analogs of heparin such as heparan sulfate, chondroitinsulfate, dermatan sulfate, dextran sulfate or any other non-toxicpolyanionic group displaying sufficient affinity for one or more of themolecular factors implicated in the wound healing process or componentssuch as monoclonal or polyclonal antibodies or microproteins that can beobtained through phage display that have a high and selective affinityfor molecular factors implicated in the wound healing process. Whenapplied onto a wound, such affinity GDP matrices have the potential toact as a reservoir for the accumulation and stabilisation of locallyavailable endogenous growth factors or other wound repair stimulatingfactors. These factors may subsequently be gradually released, thuspromoting healing of the injury. The potential of heparin-like moleculesand similar polyanions to bind and stabilize certain growth factors iswell known in the art. The following are but a few examples from thescientific literature discussing this subject. Volkin et al. havedescribed the physical stabilisation of acidic FGF by different types ofpolyanions (Arch. Biochem. Biophys., 300, p.30-41, 1993; Biochim.Biophys. Acta 1203, p.18-26, 1993). Tomoko et al. describe thestabilization of basic FGF with dextran sulfate (FEBS Letters, 306,p.243-246, 1992). Turnbull and Gallagher review the role of heparansulphate as a functional modulator of fibroblast growth factor activity(Biochem. Soc. Trans. 21, 477-482, 1993). By the incorporation of suchpolyanionic compounds in the GDP matrix of this invention, thefavourable biocompatibility and wound healing properties of the matrixmay still further be improved.

Alternatively the components incorporated into or attached to theaffinity matrix, may display an affinity for molecular factors that ishigh enough that binding can become a stable process. When applied ontoa wound, such affinity GDP matrices have the potential to specificallysequester molecular factors that are detrimental to the wound healingprocess, such as factors that cause deregulated growth or hypertrophy ora superfluous formation of collagen, and that can cause the formation ofkeloids.

In the present invention we also disclose our discovery that GDPconstitutes an efficient and versatile material for the fabrication ofslow or controlled release devices for the delivery of pharmacologicallyactive substances. It was an unexpected finding that also peptide orpolypeptide substances can be incorporated and subsequently efficientlyreleased from GDP matrices. This was surprising considering the factthat most peptides have free amino groups which can react with thealdehydes of the dextranox. One would therefore expect that thesepeptides would be irreversibly cross-linked to the GDP matrix,preventing their release. This is however not the case, as isdemonstrated in examples 6-8, showing the efficient release of anoligopeptide, iodinated IL-1α, TNF-α, IL-8, BSA and bioactive EGF. Apossible explanation for this behaviour is that the available aldehyderesidues on the dextran are out-titrated preferentially by the aminogroups of the gelatin, which is present in an about 10,000-fold higherconcentration (100 mg/ml) than the incorporated factors (present atabout 10 μg/ml).

Pharmacologically active factors of interest can be incorporated in GDPmatrices in several ways. The most preferred method is to add thefactors prior to the cross linking process. Therefore, an aqueoussolution of the active agent is mixed with an aqueous solution ofgelatin at a temperature of about 37° C., followed by mixing thissolution with an aqueous solution of dextranox. The resulting mixture isallowed to cool, during which time the gelatin sets and cross linkingbetween gelatin and dextranox chains takes place. Since gelatinsolutions are viscous, care should be taken that the differentcomponents are mixed thoroughly, so that a homogeneous distribution ofthe active agent in the GDP matrix is obtained. Another possibility isto incorporate the active factors in the GDP matrix after the crosslinking process is completed, by means of a sorption procedure.Therefore, the GDP matrix is partially or completely dehydrated. Thisdehydration can be achieved by drying the matrix in an air stream, bylyophilisation, by organic solvent extraction or by any other suitablemeans resulting in removal of water from the matrix. Subsequently, thedehydrated matrix is soaked in an aqueous solution containing theactive, agent. During this soaking process, the matrix is rehydrated, atthe same time absorbing part of the active agent.

One of the possible applications of the present invention lies in thefabrication of wound dressings containing one or more wound repairstimulating factors and/or a suitable antiseptic agent. Wound repairstimulating agents which are eligible for incorporation in such a wounddressing are for instance growth factors such as those belonging to theclass of the EGF, FGF, PDGF, TGF-β, VEGF, PD-ECGF or IGF families.Another suitable agent would be a releasate from human platelets, whichis for instance marketed by Curative Technologies Inc under the nameProcuren. Also possible would be the incorporation of a conditionedmedium, a lysate or an extract prepared from keratinocytes, such asdisclosed in patent applications U.S. Ser. No. 9,106,161 (Oregon Univ.),EP88101576 (Eisinger), WO93/10217 (IG). Suitable antiseptic agentsinclude antibiotics, antibacterial sulfamides or peptides, chinolones,antimycotics, etc., as far as they are suitable for topical use. Wounddressings containing wound repair promoting agents can be used for thetreatment of wounds which are difficult to heal. Injuries which areeligible for such treatment include but are not limited to chroniculcera, corneal injuries, tympanic membrane perforations, surgicalwounds, skin graft donor sites, burn wounds, etc. In the case of burnwounds, the wound dressings can be directly applied on a second or thirddegree burn. However, in case of extensive third degree burns, it ispreferable to first graft the burn with meshed autologous skin.Application of the medicated GDP wound dressing directly on top of thisautologous meshed graft will stimulate the closure of the meshed graftinterstices, resulting in faster wound closure and concomitant reductionof infection risks and shortening of treatment time.

To facilitate application on the treatment site, the medicated GDP wounddressings can be manufactured in different forms. For instance, sheet-or film-like dressings can conveniently be applied onto burn wounds,shallow ulcers, skin graft donor sites and other types of shallowwounds. To reduce fluid evaporation and dehydration of the dressing andthe underlying wound, the dressing can be covered with a flexiblemembrane, the water permeability of which is chosen so as to obtain anoptimal moisture level of the wound. It is also possible to manufacturemulti-layered GDP laminates. Each layer of such a laminate can havedifferent release properties and contain a different active substance.Upon application on the wound this will result in the controlled releaseof the incorporated factors from the subsequent layers, according to apredefined sequential and temporal programme. This programme will dependin part on the release properties and biodegradation of the differentlayers, their thickness and on the properties of the incorporatedfactors. Obtaining such a controlled delivery of multiple drugs isconsidered desirable because it is known that the wound repair processoccurs in different stages, each of which requires the involvement ofdifferent factors. For instance, one stage of wound healing consists ofthe development of granulation tissue. This phase may be stimulated forinstance by administration of PDGF or FGF. In a next phase, the wound isclosed by an epithelialization process, which may be stimulated by EGF.Inclusion of factors such as VEGF or PD-ECGF may optimize a process suchas vascularisation which is often unsatisfactory and can be theunderlying cause in chronic wounds such as ischaemic wounds. Whichfactor has to be released at which time point to obtain optimal healingresults, depends partly on the type of wound. It is also known thatsometimes the wound healing process can be aberrant leading to theformation of persistently heavy scars or keloids. Such keloid formationis predisposed by two main factors. The first is the location of thescar and the second is the genetic background of the patient. It istherefore anticipated that keloid formation results from the atopic orsuperfluous presence of certain factors and that the presence of certainlayers within the wound dressing can be used to sequester these unwantedfactors. Other factors that can be sequestered comprise those that canlead to superfluous production of to many collagen and/or elastin,thereby preventing phenomena such as skin contractions or keloidformation. It is also one of the advantages of the present inventionthat programmed delivery of several drugs is possible using only onedressing, i.e. without having to change wound dressings.

In case of deeper wound cavities, such as some types of pressure soresor chronic ulcers, it may be more convenient to fabricate the medicatedGDP wound dressing in the form of microparticles, foams, pastes or otherforms which are easily conformable to the wound shape. Microparticlesmay be fabricated according to any of the procedures known in the art,provided the activity of the incorporated active substances is notdestroyed. To increase the shelf life of the medicated particles, it isalso possible to lyophilize them. The resulting powder or granulate canbe applied onto the wound either directly, in which case it will havethe added benefit of adsorbing excess wound fluid, or it can be firstrehydrated by incubation in a suitable aqueous solution. The medicatedparticles can also be formulated in a suitable excipient such asvaseline, paraffin oil, etc. so as to obtain a paste which can forinstance be used to fill a cavity.

In one of the embodiments of the present invention, thepharmacologically active substance is incorporated into an affinity GDPmatrix such as described above. In this case, the matrix contains, apartfrom gelatin, dextranox and an aqueous medium, also additionalcross-linked, non-diffusible or otherwise immobilized compounds whichhave an affinity for the active substance. This results in a reductionof the release rate of the active agent and in some cases they may alsostabilize the agent. Following are but a few examples of such affinityligands which may be incorporated into GDP matrices.

One class is constituted by those molecules which display an affinityfor heparin binding proteins, such as heparin or functional analogs ofheparin such as heparan sulfate, chondroitin sulfate, dermatan sulfate,dextran sulfate or any other non-toxic polyanionic group displayingsufficient affinity for an incorporated heparin-binding factor. Examplesof such factors include FGFs, HB-EGF, amphiregulin and betacellulin.

Another example of affinity ligands may consist of hydrophobic chains,which could retard the release of incorporated active agents with ahydrophobic nature. Incorporation of such chains in GDP could beachieved for instance by the use of partially hydrophobized dextranoxderivates as cross linkers. These can be obtained for instance bypartial esterification of dextran with fatty acids (e.g. caproic acid,stearic acid) followed by periodate oxidation of the thus obtaineddextran esters.

It will be clear to the person skilled in the art that the fabricationof medicated wound dressings with controlled release properties is butone application of the present invention. Many other possibleapplications of the use of GDP as a controlled release matrix can beenvisaged. The following possibilities are intended only as examples anddo not in any way limit the range of possible applications.

GDP can for instance be used for the fabrication of devices fortransdermal drug delivery. A GDP patch containing a transdermallydeliverable drug can be attached to the skin, enabling a slow release ofthe drug over a prolonged time period. In another application, GDPmicroparticles loaded with a particular drug can be injectedintravenously, subcutaneously or intramuscularly. Equipped with atagging system, such injected microparticles may be used for topicaladministration of compounds with which the microparticles were loaded.In principle, all drugs for which a slow release over a period rangingbetween a few days to a few weeks is desirable are eligible forincorporation in GDP microparticles. Examples include, but are notlimited to, anticancer drugs, hormones, vaccines, contraceptives,cardiovascular drugs, neuroactive drugs etc.

FIGURE LEGENDS

FIG. 1: Fluid loss of GDP films covered with either an Exkin or a Pebax1205 film.

FIG. 2: Histological section of full thickness pig wound, 20 days afterapplication of GDP film, showing limited presence of inflammatory foci.

FIG. 3: Histological section of full thickness pig wound, 20 days afterapplication of Duoderm, showing presence of many granulomas.

FIG. 4: Histological section of full thickness pig wound, 20 days afterapplication of Duoderm, showing presence of vacuoles containing remnantDuoderm particles

FIG. 5: Release of AR1 oligopeptide, as measured in an elution system.AR1 peptide was incorporated during the production of the film.

FIG. 6: Release of AR1 peptide, as measured in a paper wick system,mimicking the wound situation.

FIG. 7: Release of iodinated test factors BSA, mIL1α, TNFα, and hlL-8from a 2 day-old GDP film, measured in a paper wick release system.

FIG. 8: Release of iodinated test factors BSA, mIL1α, TNFα, and hlL-8from a 1 week-old GDP film, measured in a paper wick release system.

FIG. 9: Release of iodinated test factors BSA, mIL1α, TNFα, and hlL-8from a 5 weeks-old GDP film, measured in a paper wick release system.

FIG. 10: Release of active EGF from a GDP film, measured in a paper wickrelease system.

FIG. 11: Release of polyanions from hydrogel affinity matrix samples

A) Five days after the hydrogel production

B) Two months after the hydrogel production

FIG. 12: Temperature dependence of the hydrogel storage modulus infunction of hydrogel storage time and hydrogel dextran sulphate content.

FIG. 13: Water uptake by dextran sulphate-containing gelatin hydrogelsat 20° C. and 37° C.

FIG. 14: Water uptake at 37° C. by gelatin hydrogels containingdifferent concentrations of dextran sulphate

FIG. 15: Wound healing in pig

A) Reduction of the initial wound area by wound contraction andre-epithelalization

B) Reduction of the initial wound area by wound contraction

FIG. 16: Wound healing in pig

A) Reduction of the initial wound perimeter by wound contraction

B) Reduction of the initial wound perimeter by wound contraction andre-epithelialization

FIG. 17: Rate of wound healing

Radial progression towards wound closure: length of daily advance of thewound margins and the re-epithelialization towards wound center

EXAMPLES Example 1 Production of GDP Film

Oxidation of Polysaccharide

Twenty g of pyrogen-free clinical grade dextran with molecular mass60,000-90,000, such as commercialised by ICN (ref:101513) are dissolvedin 200 ml of phosphate buffer (pH6). A solution which is prepared from7.92 g of periodate and 20 ml water is subsequently added, withagitation, to the dextran solution. After two hours at room temperature,the oxidized polysaccharide is isolated from the reaction medium byprecipitation with a third solvent. Before precipitation, iodate formedduring the reaction can be eliminated by treating the reaction solutionwith 30 g of potassium iodide dissolved in 150 ml of dilutedhydrochloric acid (0.05M), before pouring the mixture into 500 ml ofmethanol to precipitate the oxidized dextran.

Alternatively, the reaction mixture can be dialysed against pure waterand, when all the periodate is eliminated, the solution can beconcentrated to isolate the polysaccharides. Concentration can beperformed for instance by lyophilisation. It has been established bytitrimetric analysis using hydroxylamine that 20% of the secondaryalcohol groups of the dextran were oxidized.

Preparation of a GDP Film

Two g of gelatin (Isoelectric point 7.01, gel strength 203 on the Bloomscale) is dissolved at 40° C. in 10 ml of phosphate buffer (pH 8, ionicstrength 0.2). Ten g of dextranox prepared as describe above isdissolved in 10 ml of phosphate buffer (pH 8, ionic strength 0.2) andwarmed to 40° C. Both solutions (at 40° C.) are mixed and subsequentlypoured into a cast made of two glass plates separated by spacers of 1 mmthickness. The cast is then placed overnight at 4° C. during whichgelation and cross-linking take place. After removal of the glass platesa flexible 1 mm thick film is obtained which is water-insoluble.

Example 2 Preparation of a GDP Film Laminated to a Plastic Foil

Preparation of a GDP Film Laminated to a Plastic Foil

Two 9 of gelatin (Isoelectric point 7.01, gel strength 203 on the Bloomscale) is dissolved at 40° C. in 10 ml of phosphate buffer (pH 8, ionicstrength 0.2). Ten g of dextranox prepared as describe above isdissolved in 10 ml of phosphate buffer (pH 8, ionic strength 0.2) andwarmed to 40° C. Both solutions (at 40° C.) are mixed and subsequentlypoured into the reservoir of a film casting device (Braive Instruments).The casting plate of the film casting device has been fitted beforehandwith a 20 μm thick plastic film of the type Pebax X 1205 (available fromElf Atochem, France). The sliding knife and reservoir of the castingdevice is set at the desired film thickness. The casting plate of thedevice is thermostatized at 15° C. to shorten the gelling time of thecasted polymer mixture and thus ensure even film thickness. Film castingis performed by moving the sliding knife and reservoir with a presetspeed (for instance 2 mm/sec.) over the casting plate, thus leaving athin polymer film onto the plastic foil. After casting the GDP film ontothe Pebax foil, it is allowed to gel and cross link overnight at 4° C.The result is a GDP film firmly laminated onto the 20 μm thick Pebaxfoil. Alternatively, depending on the desired water vapour permeability,another type of plastic foil can be used, for instance Exkin (producedby Utermohlen NV, The Netherlands). This plastic foil, which can be usedas a semi-occlusive wound dressing, has a bilayer structure consistingof a macroporous and a microporous layer. Due to its higher porosity, ithas a much higher water vapour permeability as compared with the Pebax X1205 foil.

Comparison of the Water Loss Due to Evaporation of GDP Films Laminatedto Different Plastic Foils.

A 1 mm thick GDP film is laminated to either a 20 μm thick Pebax X 1205foil or to an Exkin foil and placed with the GDP side down onto a metalplate heated to 37° C. The upper side of the laminate (i.e. the plasticfoil) is exposed to the air at ambient temperature (approximately 20°C.). At regular time points, water loss of the films is measured bydetermining the residual weight of the laminates. This residual weightis expressed as percentage of the original weight and is displayed inFIG. 1. From the results, it is clear that the Pebax X 1205 foil has abetter barrier function and may thus be suited for the preparation ofGDP laminate wound dressings intended for the treatment of woundsproducing low amounts of exudate. On the contrary, the Exkin membraneallows a higher evaporation rate and is consequently more suited forpreparation of GDP laminate wound dressings intended for the treatmentof wounds producing high exudate volumes.

Example 3 In Vitro Cytotoxicity Testing

One of the most important prerequisites for the clinical usefulness of awound dressing is that it has a high biocompatibility. Therefore, it isessential that the material displays a very low or even non-existentcytotoxicity. Cytotoxicity of a biomaterial can be measured in vitro byincubating the material for a prolonged period together with suitabletarget cells. If the material is cytotoxic, the target cells will bekilled and the number of surviving cells will be inversely related tothe cytotoxicity. This can conveniently be done in a so-calledmethylcellulose toxicity test, as described by Van Luyn (Doctoralthesis, University of Groningen, The Netherlands, 1992;ISBN-90-9005113-9). As target cells, different cell types relevant forwound healing can be used, such as fibroblasts, keratinocytes andendothelial cells.

Cytotoxicity of GDP Film for Murine 3T3 Fibroblasts

Twenty five thousand Swiss 3T3 fibroblasts are seeded per well in12-well tissue culture plates. The cells are seeded in standard growthmedium containing 1.125% methylcellulose. The cells are allowed toattach for 24 hours at 37° C. Subsequently, a 113 mm² circular piece ispunched from a GDP film and placed on top of the methylcellulose gelcovering the seeded cells. For comparison a similarly sized piece of thehydrocolloid ulcer dressing Duoderm (obtained from Convatec, UK) isplaced on another well. A third well serves as negative control andreceives no test material. All tests are carried out in triplicate.After 6 days at 37° C., the amount of surviving cells is determined byMTT staining, a method which specifically detects metabolically activecells. The percentage of surviving cells (relative to the negativecontrol) is 22% and 42% with Duoderm and GDP, respectively. Thisindicates that GDP has a considerably lower cytotoxicity towards thesefibroblasts than the commonly used ulcer dressing Duoderm.

Cytotoxicity of GDP Film for Human Skin Fibroblasts

Twenty five thousand primary fibroblasts isolated from human skin areseeded per well in methylcellulose-containing medium in 12-well tissueculture plates as described above. After 24 hours at 37° C., circularpieces of Duoderm or GDP are applied on top of the methylcellulose gel.The GDP films used in this experiment have been sterilized by gammairradiation. Two irradiation doses are used: one film receives 2.5 MRad,another film receives 0.6 MRad. The pieces are left in place for 6 days,after which the percentage of surviving cells relative to a negativecontrol is determined using MTT staining. The percentage of survivingcells is 63, 59 and 44% relative to the negative control for GDPirradiated with 2.5 MRad, GDP irradiated with 0.6 MRad and Duoderm,respectively. Again, these figures demonstrate that GDP film isconsiderably less cytotoxic for fibroblasts than Duoderm. Moreover,irradiation does not induce cytotoxicity in the GDP films.

Cytotoxicity of GDP Film for Murine Balb/MK Keratinocytes

Twenty five thousand murine Balb/MK keratinocytes are seeded per well inmethylcellulose-containing medium in 12-well tissue culture plates asdescribed above. After 24 hours GDP film and Duoderm are applied ontothe methylcellulose gel and left in place for eiher 3 or 6 days. After 3days, the percentage of surviving cells is 65 and 30% with GDP andDuoderm, respectively. After 6 days of incubation with the wounddressings, the percentage of surviving cells is 32 and 9% with GDP andDuoderm, respectively. This again confirms the superior cytotoxicityproperties of GDP above Duoderm.

Cytotoxicity of GDP Film for Human Skin Keratinocytes

Twenty five thousand primary human keratinocytes isolated from a skinbiopsy are seeded per well in hydroxyethylcellulose-containing medium in12-well tissue culture plates as described above. In this case,hydroxyethylcellulose (marketed under the trade name Idroramnosan byVevy, Italy) is used instead of methylcellulose because we have foundthe former material to be more suitable to support the attachment andproliferation of the human keratinocytes. After 24 hours at 37° C.,pieces of GDP film and Duoderm are placed onto the gel and left in placefor 3 or 6 days. After three days, the percentage of surviving cells is79% and 13% with GDP and Duoderm, respectively. After 6 days, 35 and1.4% of the cells survive with GDP and Duoderm, respectively. Once more,this underscores the superior properties of the GDP dressing, since ithas only a limited cytotoxicity for keratinocytes, while incubation withDuoderm results in almost 100% cell death within 6 days.

Conclusions on the In Vitro Cytotoxicity Testing

The in vitro cytotoxicity tests described above show that GDP has a veryfavourable and low cytotoxicity level. We have compared GDP with Duodermbecause both dressings are of a similar type and because the latter is avery frequently used dressing for the treatment of chronic ulcers. Thefact that GDP is superior to Duoderm with respect to cytotoxicityunderscores its clinical applicability as a wound dressing.

Example 4 In Vivo Biocompatibility Testing: Implantation in Mice

GDP film of 1 mm thickness is prepared as described in example 1. GDPstrips of 2×30 mm are implanted subcutaneously on the back of Balb/Cmice. Per mouse, two strips were implanted bilaterally, parallel withthe spine, under the panniculus carnosus. At 1, 2, 4, 8 and 16 daysafter implantation, the implantation sites are controlledmacroscopically and then completely excised. Per time point, two miceare used; Excised sites are processed for histology and evaluatedmicroscopically. As controls, similarly sized strips of Duoderm areimplanted in a separate set of mice, and in yet another set of micesubcutaneous pockets are made which receive no implant.

Macroscopically, slight redness is observed with Duoderm. New tissue isfound to invade the material. Duoderm, however, stays largely intact.With GDP, no macroscopically visible reactions are observed and thematerial stays intact.

Histologically, Duoderm shows slight inflammation on day 1, withincreasing infiltration of neutrophils from day 2 onwards. At days 4 or16, strong inflammation is observed with infiltration of neutrophils andmacrophages. Lipid droplet-containing macrophages are present and theinflammation is more of the chronic type, with some signs of foreignbody reaction.

With GDP, a strong neutropbil infiltration is also observed initially.However on day 16, the inflammatory response largely disappears. Nosigns of foreign body reaction are observed. On days 8 and 16, slightlyincreased fibroblast proliferation is observed around the implant. TheGDP implant remains almost completely intact, apart from some slighterosion at the edges.

These results confirm the biocompatibility of GDP. The quite normalinflammatory response initially observed resolves rapidly, and no signsof long term inflammatory events or foreign body reactions are observed.This means the material is well suited for the fabrication of wounddressings.

Example 5 In Vivo Testing: Full Thickness Wounds in Pigs

Twenty-four full thickness square wounds of 2×2 cm are made on the backand sides of a 75 kg castrated male Belgian Landrace pig. The wounds aremade under general anaesthesia (Stresnil, Halothane and Diprivan), byexcising the skin to the fascia, taking care to include no muscle withthe excised tissue. Each side of the animal receives two rows of 6wounds, parallel to the backbone. After surgery, 8 wounds are coveredwith a 5×5 cm sheet of GDP film of 1 mm thick, prepared as described inexample 1. Eight other wounds are treated with a 5×5 cm sheet ofDuoderm, while the remaining eight wounds serve as controls. All woundsare subsequently covered with Tegaderm (an occlusive polyurethanedressing) and fixed with Fixomull and Velpo bandages. At 2, 5, 9 and 20days after surgery, two wounds of each treatment are examinedmacroscopically and subsequently fully excised for histologicalanalysis. Epithelisation and contraction of the wounds arequantitatively evaluated by planimetry. Macroscopically, both GDP andDuoderm appear to stimulate granulation tissue formation on days 5 and 9after surgery, with the wound bed protruding several mm above thesurrounding skin. Both GDP and Duoderm result in increasedepithelisation on day 9. At day 20 after surgery, all wounds are closedand no gross macroscopical difference between the three treatments isobserved. For histological analysis, wound biopsies are fixed in 10%paraformaidehyde, sectioned at 5 μm and stained either withhaematoxylin/eosin or with Masson's Trichrome. The progression of theepithelial margin is measured on both sides of each wound, using tworepresentative tissue sections. The results are expressed in Table 1 andindicate a slightly increased epithelisation with GDP on day 5, while onday 9, the difference between the three treatments was non-significant.On day 2 after surgery, the general appearance of all wounds is similar,although Duoderm-treated wounds contain more red and inflammatory cellsand several inflammatory foci of polymorphonuclear neutrophils andmacrophages/monocytes. At that time point, GDP is still largely intact.At five days after surgery, those parts of GDP which are in contact withthe tissue are strongly invaded by white cells while the parts incontact

                  TABLE 1                                                         ______________________________________                                        Epithelialisation rates of full thickness pig wounds treated with GDP,        Duoderm or Tegaderm, measured directly on histological sections (a) or        on micrographs of histological sections (b). Significance of the results      is                                                                            displayed in the second table.                                                        Days after wounding                                                   Treatments                                                                              2          5           9                                            ______________________________________                                        GDP     a     212.3 ± 227.3                                                                         1898.0 ± 736.9                                                                       2087.2 ± 203.7                                  b     197.3 ± 160.7                                                                         1779.4 ± 271.4                                                                        1974.4 ± 332.48                        Duoderm a     415.0 ± 297.8                                                                         1651.0 ± 523.5                                                                       2578.5 ± 473.8                                  b     360.6 ± 166.7                                                                         1415.7 ± 445.8                                                                       2504.8 ± 240.7                          Tegaderm                                                                              a     651.6 ± 278.2                                                                         1091.5 ± 276.3                                                                       2215.2 ± 384.4                                  b     613.9 ± 427.2                                                                          844.9 ± 403.5                                                                       2202.6 ± 370.9                          ______________________________________                                        Analysis of the variance                                                      ANOVA (F-test, Bonferroni method), NS: not significant, S : significant       at 95%, SS significant at 99%.                                                                 Days after wounding                                                                 2         5   9                                        ______________________________________                                        GDP versus Duoderm                                                                             a     NS        NS  S                                                         b     NS        NS  SS                                       GDP versus Tegaderm                                                                            a     S         S   NS                                                        b     S         SS  NS                                       Duoderm versus Tegaderm                                                                        a     NS        NS  NS                                                        b     NS        NS  NS                                       ______________________________________                                    

with crust or fibrin clot are nearly intact. In Duoderm-treated wounds,a dense inflammatory reaction is seen, including the formation ofgranulomas. At day 9 after surgery, GDP-treated wounds are filled withgranulation tissue above the level of the surrounding skin. The GDPmaterial in contact with tissue is totally invaded by white cells, whilethe crust-contacting GDP is mostly intact. Some granulomas are present,but much less than with Duoderm. The deeper dermal tissue contains fewinflammatory cells and consists of dense scar tissue with regularlyarranged fibroblasts embedded in dense collagen. In Duoderm-treatedwounds, a similar scar tissue is present, but the granulomas are by farmore abundant than with GDP (FIGS. 2 and 3). Tegaderm-treated woundscontain almost no granulomas. At 20 days post-surgery, all wounds areepithelialized. Granulomas are very abundant in Duoderm-treated wounds,much less abundant in GDP-treated wounds and only occasionally seen inTegaderm-treated wounds. Scar tissue of Duoderm-treated wounds showsmany vacuoles containing remnant Duoderm particles (FIG. 4). Thisindicates a strong, moderate and weak inflammatory foreign body responsewith Duoderm, GDP and Tegaderm, respectively. The conclusion of thistest is that GDP is a highly biocompatible material, which generates asignificantly lower long term inflammatory response than the widely usedulcer dressing Duoderm. Moreover, the material is completelybiodegradable over a period of 1-3 weeks, although it remains largelyintact for about 5 days following application.

Example 6 Controlled Release of an Oligopeptide from GDP Films

Preparation of a Polypeptide-loaded GDP Film

Two g of gelatin (Isoelectric point 7.01, gel strength 203 on the Bloomscale) is dissolved at 40° C. in 10 ml of phosphate buffer (pH 8, ionicstrength 0.2). One g of dextranox prepared as described by Schacht etal. (Pat. appl. 0308330) is dissolved in 10 ml of phosphate buffer (pH8, ionic strength 0.2) and warmed to 40° C. To both solutions, 0.1%thimerosal is added as a preservative. To 10 ml of the gelatin solution,100 μg of a biotinylated 21-mer oligopeptide (AR1) corresponding to theN-terminal part of the growth factor amphiregulin is added. The AR1oligopeptide has the following amino acid sequence:Biotin-GG-VKPPQDKTESENTSDKPKR-CONH₂. Biotinylation of this oligopeptidewas carried out only to facilitate its subsequent quantization but isotherwise not relevant for the result of the release experiments.Against this peptide a polyclonal antiserum raised in rabbits isavailable (antiserum RB 425), which allows detection of the peptide inan ELISA test system with a sensitivity of 2 ng/ml. Both the dextranoxand peptide-containing gelatin solutions (at 40° C.) are mixed andsubsequently poured into a cast made of two glass plates separated byspacers of 1 mm thickness. The cast is then placed overnight at 4° C.during which gelation and cross-linking take place. After removal of theglass plates a flexible 1 mm thick film is obtained which iswater-insoluble. From this film, circular discs with a surface area of400 mm² were punched for performing the release studies.

In Vitro Release Studies

Release Testing Using an Elution System

Circular AR1 peptide-containing GDP discs with a volume of 400 μl arebriefly rinsed in Phosphate Buffered Saline (PBS) and incubated withslight agitation (approx. 60 rpm on an orbital shaker platform) in 4 mlof PBS containing 0.1% casein. The release test is carried out at anambient temperature of 22° C. After 0, 1, 2, 4, 8, 24, 48 and 96 hoursof incubation, 300 μl of the incubation solution is removed, snap-frozenin liquid nitrogen and stored at -20° C. After each sample removal, 300μl of fresh PBS-casein buffer is added to the incubation vessel tomaintain a constant extraction volume. After completion of the elutions,the samples are thawed, diluted 1/1, 1/10 an 1/100 and the amount of AR1peptide is quantitated in an AR1-ELISA using the AR1-specific RB425antibody as the first antibody and an alkaline phosphatase-conjugatedanti-rabbit second antibody. The obtained results are shown in FIG. 5.The amount of AR1 released is expressed as percentage of the originalamount of AR1 which is incorporated in the GDP disc. It can be seen thatall the incorporated peptide is released within approximately 4 hours.This demonstrates that, despite the presence of 4 lysines in thepeptide, no appreciable irreversible cross-linking of the peptide to thematrix has taken place.

Release Testing Using a Wound-mimicking System

For evaluation of release kinetics of controlled delivery wounddressings, an elution test system as described above is not ideal. Sincethe elution is carried out by means of an active extraction procedure,the release in such a system is much faster than would be observed in awound. Therefore, we have adopted an alternative test system whichmimics the wound situation with greater accuracy. This system has alsobeen described by Shinde and Erhan (Bio-Med. Mat. Eng. 2: pp. 127-131,1992) for determining the release properties of insulin-loadedflexibilized gelatin films. In this system, circular GDP discs (400 μl)are placed on a wick which is on its turn placed in a petri dishcontaining 4 ml of extraction fluid (PBS containing 0.1% casein). Thewick is constructed of either 2 or 5 stacked Whatman 3MM filter paperdiscs with the same diameter as the GDP test slice. In case of a wickwith 2 Whatman discs, the top surface of the wick is level with thesurrounding extraction fluid. In case of a wick with 5 Whitman discs,the top surface of the wick is slightly higher than the level of theextraction fluid. The release test is carried out at 22° C. Afterplacing the AR1-containing GDP test slice on the wick, the petri dish isclosed and 300 μl samples are removed from the extraction fluid atregular time points as described above. AR1 present in the samples isquantitated in an AR1-specific ELISA system as described above. Theresults are shown in FIG. 6. In this example, between 45% and 50% of theincorporated AR1 peptide is released, over a time period ofapproximately 5 days. The fact that no 100% release is obtained ispartly due to retention of some AR1 in the wick. Nevertheless, therelease of AR1 in this system can be considered as highly efficient, andwith a kinetics profile which is suitable for application in a wounddressing.

Example 7 Controlled Release of Iodinated Test Polypeptides (IL-1α,IL-8, EGF, BSA) from GDP Films

Preparation of the Films

For application in the manufacturing of growth factor-containingmedicated wound dressings, GDP should also allow the efficient releaseof larger peptide factors. To evaluate this, a number of test proteinswith different properties is used. To facilitate the release studies,these proteins are iodinated prior to their incorporation. The testproteins used are listed below:

                  TABLE 2                                                         ______________________________________                                        protein  MW (kDa)  GRAVY       pl   % lysine                                  ______________________________________                                        BSA      69        -4.3        5.69 10.7                                      IL-1α                                                                            17        -1.65       5.09 9.2                                       TNF-α                                                                            17        -2.09       7.26 12.8                                      IL-8     8         -4.88       9.59 12                                        ______________________________________                                    

Iodization is performed according to the iodobeads method (Pierce, US).The specific activity obtained ranges between 3000 and 8000 cpm/ng ofprotein.

GDP films containing the iodinated factors are prepared using similarprocedures as described above for AR1-containing films, except that thegelatin solution is presterilized by autoclaving. The concentration ofiodinated test proteins in the GDP matrix is approximately 8 μg/ml.

Release Tests

The release tests are set up using the wick system as described abovefor AR1, using a wick containing 5 stacked Whitman 3MM filter discs. Tosimulate the wound conditions more closely, the release test is carriedout in a thermostatized incubator at 37° C. At predefined time points,100 μl extraction fluid is removed and 100 μl fresh fluid is added tomaintain a constant extraction volume. To quantitate the amount oflabelled protein released, the radioactivity present in the removedextraction liquid samples is measured in a gamma-counter. Toadditionally evaluate the stability upon storage of the protein-loadedGDP films, release profiles are determined in films stored at 4° C. for2 days, 1 week and 5 weeks. Previous experiments have shown that, aftera storage time of the labelled proteins of 52 days at -20° C., up to 25%of the incorporated iodine could be released from the proteins due toradiolysis. Therefore, extraction liquid samples are first precipitatedwith TCA prior to the radioactivity measurements, to be sure that onlyprotein-associated radioactivity is quantified. At the end of theexperiment, residual radioactivity is also determined in the GDP discsand in the filter paper wick.

The results are shown in FIGS. 7-9. Approximately 50-80% of theincorporated proteins are released in a period of 6 days. The amount ofresidual protein in the GDP disc and the wick ranged from about 10 toabout 22%. The results confirm that also for larger proteins, releaseoccurs with high efficiency and according to kinetics which arefavourable for application in medicated wound dressings. Also, thestability of the matrix proves to be sufficient to allow prolongedstorage.

Example 8 Controlled Release of Bioactive EGF From GDP Films

The potential of GDP films to release oligopeptides and proteins is wellestablished, as demonstrated in examples 1 and 2. For effective use, itis however also important that the biological activity of the releasedfactors is preserved. To demonstrate that this is indeed the case, a GDPfilm is produced containing the growth factor EGF. EGF stimulates a widevariety of cell types, including keratinocytes and fibroblasts, and isgenerally accepted as a suitable candidate factor for stimulating thewound repair process. Several companies are actively involved indeveloping EGF for therapeutical purposes. Therefore, EGF can beregarded as an appropriate molecule to be incorporated in medicatedwound dressings such as those disclosed in the present invention.

Preparation of GDP Film Containing EGF

Mouse submaxillary gland EGF is obtained from Sigma (US).

GDP film containing EGF is prepared according to the procedure describedabove for AR1. The gelatin used for the preparation of the films hasbeen sterilized by a 0.6 MRad gamma radiation dose before dissolution.The final concentration of EGF in the film is approximately 10 μg/ml.The EGF-containing GDP film is stored at 4° C. for 1 week until use inthe release test.

Release Experiment

To simulate release under wound-like conditions, a new test system isset up. A disc of 113 mm² is punched from the GDP film and placed on thefilter membrane of a Trans-well filter cup (Costar, US) with a filterpore size of 3 μm. The cup is subsequently placed in a well from a6-well test plate. The well contains 1 ml of extraction fluid consistingof PBS containing 0.05% CHAPS and 0.1% casein. The Transwell cup isinserted in such a way that the membrane just touches the surface of theextraction fluid. After assembling the system, the 6-well plate isclosed with a lid and incubated at 37° C. in a thermostatized incubator.At selected time points, 50-μl samples are removed from the extractionfluid and frozen at -70° C. After each sampling, fresh extraction fluidis added to the well in order to maintain a constant liquid volume. Atthe end of the test, all samples are thawed and the amount of EGF isquantitated using a bioassay. In this bioassay, EGF or test samples areadded to a culture of growth-arrested Balb/MK keratinocytes. Uponstimulation with EGF these cells increase their proliferation rate. Thisproliferation rate can in turn be quantitated by measuring the amount oftritium-labelled thymidine incorporated in the DNA of the cells. Aftercomparison of the tritium thymidine incorporation values of test sampleswith the values obtained using known concentrations of EGF, the amountof active EGF in these test samples can be accurately determined.

The results of the release test are shown in FIG. 10. More than 60% ofthe incorporated EGF is released over a period of about 7 days. Thisexample effectively demonstrates that a wound repair-promoting growthfactor can be incorporated in GDP and subsequently efficiently releasedin a bioactive form. GDP matrices according to this invention aretherefore suitable controlled release devices for the fabrication ofmedicated wound dressings.

Example 9 SIPN Incorporation Technology and Release Experiments

SIPN Incorporation Technology

The GDP hydrogel films are prepared as demonstrated in example 1.Polyanions are incorporated into the hydrogel matrices during theproduction before gelatin hydrogel cross-linking to result in a SIPN(semi-interpenetrating polymer network). The hydrogel matrices filmsconsist of 5% dextran dialdehyde (20% oxidized dextran, MW 70000), 10%gelatin type A and one of the following polyanions: chondroitin sulphate(MW 15000), dextran sulphate (MW 40000 or 400000-600000), at a finalconcentration of 0.5% (5 mg/ml) or heparin (at a final concentration of200 μg/ml). To achieve a homogenous distribution of the incorporatedpolyanions in the hydrogels, the polyanions are first mixed in thedextran dialdehyde solutions before mixing the polyanion-containingdextran sulphate solutions with gelatin solutions. The polyanions usedare:

Dextran sulphate (MW 400000-600000), ICN Biochemicals, Cleveland, Ohio,Lot N° 64914.

Dextran sulphate (MW 40000), ICN Biochemicals, Cleveland, Ohio, Lot N°66617.

Chondroitin sulphate (MW 15000), RUG

Heparin sodium salt from Porcine intestinal mucosa sodium salt Grade I175 USP/mg Sigma Chemical Company St Louis, Mo. Lot N 63H1017. (MW6000-20000).

Heparin sodium salt from Porcine intestinal mucosa sodium salt Grade I175 USP/mg Sigma Chemical Company St Louis, Mo. Lot N 21H7705. (MW4000-6000).

The affinity matrix films are stored at 4° C. for either 5 days or twomonths in sealed plastic packages to prevent hydrogel drying.

Release Experiments

To quantify the release of polyanions from the affinity matrix, filmsamples (3.8 cm²) are immersed in 3 ml PBS/thimerosal 0.02% andincubated at 37° C. for 7 days with agitation (1 rotation/sec). Atparticular time points, the extraction medium is removed, immediatelystored at 4° C., and replaced by fresh extraction medium. At the end ofeach experiment, the amounts of polyanions released in the medium arequantified by a calorimetric method which uses dimethylmethylene blue(Farndale et al BBA 883: 173-177, 1986).

The release kinetics of polyanions from cross-linked gelatin hydrogelsstored up to 2 months at 4° C. are evaluated. FIGS. 11 (A and B) showsthe appearance of the polyanions in the extraction medium during the 7day incubations. During incubations of 5 day old hydrogels (FIG. 11A),polyanions of low molecular weights (heparin and chondroitin sulphate)are completely released from the affinity matrix samples within a fewhours. The majority (>80%) of dextran sulphate (MW: 40000) is releasedwithin 2 day incubations, this burst release is followed by a plateaurelease during the next 5 day incubation, leading to the completepolyanion release. By contrast, only about 30% of thehydrogel-incorporated dextran sulphate (MW: 400000-600000) is releasedduring the 7 day incubations. A similar release experiment (FIG. 11B)performed 2 months after the hydrogel production showed similar patternsfor polyanions of low molecular weights (heparin and chondroitinsulphate), but the released amounts of dextran sulphate (MW: 40000 and400000-600000) are markedly decreased to about 44 and 10%, respectively.This example thus indicates that in order to use the affinity matrix asa reservoir for endogenous growth factors, polyanions of low molecularweight have to be cross-linked to the hydrogel matrix. Affinity matricescontaining polyanions of high molecular weight (dextran sulphate MW400000-600000) can be used without additional cross-linkage of thepolyanions, since after the process of chemical cross-linking andphysical structuring of the gelatin hydrogel matrix which takes aboutone week, only about 10% of the hydrogel matrix incorporated polyanionis released.

Example 10 Swelling and Visco-elastic Properties of the DextranSulphate-containing Cross-linked Gelatin Hydrogel Films

Preparation of Dextran Sulphate-containing Affinity Matrices.

Gelatin type B (G-9382, lot 26H0347) from Sigma is prepared by alkalinetreatment of bovine skin. The gel strength is 225 Bloom. The gelatin issterilized by gamma-irradiation (0.6 Mrad) prior to use. Dextran (MW70000) purchased from Pharmacia Fine Chemicals (Uppsala, Sweden) isdried at 80° C. on phosphorus pentoxide. Dextran dialdehydes (20%oxidized dextran) are prepared as described in example 1. Dextransulphate (MW500000, sulphur content 17%) is obtained from Pharmacia FineChemicals (Upsala, Sweden). The hydrogels are prepared by physically(SIPN) entrapping dextran sulphate during the cross-linking of gelatinand dextran dialdehydes. The gelatin and dextran dialdehydeconcentrations are 10 and 5%, respectively, and four concentrations ofdextran sulphate (0, 0.1, 0.5 and 1wt %) are incorporated in thehydrogel matrix. The reaction is performed in an aqueous medium(phosphate buffer, pH 6.5). Both solutions are mixed and the finalsolutions are stirred at 40° C. for 1 minute. The 1 mm hydrogel filmsare prepared as described in example 1.

Visco-elastic and Swelling Measurements

The Theological measurements at oscillatory shear deformation on thehydrogel films are carried out with a CSL² Rheometer (TA Instruments)using parallel rough plates of 40 mm diameter and a plate-to-platedistance of 800 μm. The temperature dependence of the storage (elastic)modulus is determined by oscillatory shear deformation and temperaturescan in the range from 16 to 50° C. (heating rate 1.75° C. min⁻¹) atconstant frequency (1 Hz) and constant shear strain (γ=0.05, 1.8 mrad).The hydrogel formation (gelation) of dextran dialdehyde-gelatin aqueoussolutions is governed by two strong interactions. One is associated withthe chemical cross-linking of gelatin and dextran dialdehydes(gelatin-dextran chemical interaction) and the other is based on theability of gelatin to form polymer 5 network structures that arestabilised by physical cross-linking (gelatin-gelatin physicalstructuring). The temperature scan of the hydrogel samples below andabove the hydrogel melting point allows the evaluation of the respectivecontribution of the chemical and the physical cross-linking interactionsin the hydrogel formation.

For measurements of the swelling properties, the cross-linked gelatinhydrogel films, prepared as described above, are stored at 4° C. for oneweek. Samples of hydrogels (1 mm thick discs, 32 mm in diameter) areweighed and then immersed in 80 ml of PBS buffer (pH 7.4) maintained ateither 20° C. or 37° C. The immersion medium contains sodium azide. Atparticular time points, the hydrogels are removed from the immersionsolution, and after quick blotting with filter paper, weighed. Eachpoint presented in this study is the mean value of triplicatemeasurements. Results are expressed as percentage of swelling (S %) andwere calculated by using the following equation:

    S%=[(W.sub.ht -W.sub.d0)/W.sub.d0 ]*100

where W_(d0) is the weight of the dry gel at time 0 and W_(ht) is theweight of the hydrated or swollen gel on time t. The swellingexperiments are started by using a series of hydrated hydrogel samples:i.e. hydrogel samples with 15 wt % of polymer content and about 600 wt %of water content (percentage of swelling). Hydrated hydrogel samples areused because drying the samples prior to the start of the experimentscauses supplementary matrix cross-linking and a concomitant decrease inthe swelling capacities of the hydrogel films. For this reason, the dryweight of the hydrogel samples at time 0 (W_(d0)) is estimated byweighing another series of hydrogel samples taken from the same hydrogelfilms. At the end of the immersion incubations, the hydrogel samples aredried on phosphorus pentoxide and weighed again to evaluate the hydrogelsample final dry weight. The loss of hydrogel dry weight during theswelling experiment allows the calculation of a hydrogel solfraction andgelfraction. The solfraction is the fraction of the polymer lost duringthe hydrogel immersion incubation and the gelfraction is the remainingpart of the polymer.

The Visco-elastic Properties of Cross-linked Gelatin Hydrogels FilmsContaining Physically Entrapped Dextran Sulphate

The effect of increasing the storage time on the visco-elasticproperties of the dextran sulphate-containing cross-linked gelatinhydrogel films is evaluated by rheological measurements. The hydrogelfilms containing four different concentrations of dextran sulphate (0,0.1, 0.5 and 1%) are stored at 4° C. during 1 day, 7 and 25 days. Thetemperature dependence of the storage modulus (G') in function ofhydrogel storage time is shown in FIG. 12. The cross-linked hydrogelfilms show a large increase in their strength with increasing storagetime, most of the physical cross-linking occurred during the first day,while the chemical cross-linking is strongly improved with longerstorage time. The hydrogels containing different amounts of dextransulphate show almost identical curves. The storage modulus G' is notsignificantly modified by the addition of dextran sulphate (up to 1%),indicating neglectable influence of those concentrations of dextransulphate on the visco-elastic properties of the hydrogel films.

Swelling Properties of Cross-linked Gelatin Hydrogels ContainingPhysically Entrapped Dextran Sulphate

The water uptake of cross-linked gelatin hydrogel films containingincreasing amount of dextran sulphate (0, 01, 0.5 and 1%) is evaluatedafter hydrogel PBS-immersion incubations at either 20° C. or 37° C. Asseen in FIG. 13, at 20° C. hydrogel water uptake reaches a plateau after3 hours of hydrogel immersion at a swelling percentage of about 900%. At20° C., the plateau values are not changed by the presence of increasingamount of incorporated dextran sulphate. At 37° C., the plateau valuesare reached later (after about 24 h of hydrogel immersion) and arehigher than those recorded at 20° C. Moreover, at 37° C., the plateauvalues are modified (increased) by the presence of dextran sulphate inthe hydrogel matrix. The effect of the presence of increasing amount ofdextran sulphate on the swelling properties at 37° C. is shown in FIG.14. After one-day incubations, hydrogel films containing no dextransulphate absorb water to about 1200%, those containing 0.1% dextransulphate show more water uptake (to about 1400%) and those containingeither 0.5 or 1% further absorb water to about 1500%. There is nofurther hydrogel water uptake by increasing the hydrogel dextransulphate content from 0.5% to 1%. At the end of the swelling incubationtime, hydrogel samples are dried and weighed and the final dry weightsare compared with the initial dry weight in order to evaluate thehydrogel loss of weight during the incubation. Table 3 gives thegelfraction and the solfraction of the hydrogels of the differentcomposition.

                  TABLE 3                                                         ______________________________________                                        Incubation                                                                              Hydrogel dextran                                                    Temperature                                                                             sulphate content                                                                            Gelfraction                                                                             Solfraction                                 (°C.)                                                                            (wt %)        (%)       (%)                                         ______________________________________                                        20        0.0           78 ± 1 22 ± 1                                             0.1           77 ± 0 23 ± 0                                             0.5           79 ± 0 21 ± 0                                             1.0           79 ± 1 21 ± 1                                   37        0.0           63 ± 1 37 ± 1                                             0.1           51 ± 9 49 ± 9                                             0.5           58 ± 3 42 ± 3                                             1.0           60 ± 4 40 ± 4                                   ______________________________________                                    

The solfractions that are find at 37° C. are higher than those found at20° C., indicating that an increased fraction of the hydrogel componentsis released from the hydrogel samples during the incubations at 37° C.The gel- and sol-fraction of all gelatin hydrogels containing differentconcentrations of dextran sulphate are comparable.

The Theological measurements shown in this example thus further confirmthat the process of chemical cross-linking and physical structuring ofthe dextran dialdehyde cross-linked gelatin hydrogel matrix does notoccur instantaneously. Here again, important changes in the mechanicalproperties occur during the first week after the hydrogel production,thereafter, the elastic properties are stabilised. The presentrheological study shows that the incorporation of dextran sulphate (upto 1%) into the hydrogel matrix does not modify the elastic propertiesof the hydrogel films.

The swelling experiments show that in the presence or the absence ofdextran sulphate in the cross-linked gelatin hydrogel films, hydrogelwater uptake is higher at 37° C. than at 20° C. Above the melting pointof gelatin (at 37° C.), the physical structuring of the hydrogel isdestroyed leading to hydrogels with a lower density and causing betterswellability of the hydrogel. When the physical structuring of thehydrogels are destroyed (at 37° C.), the presence of dextran sulphatecontributes to increase the hydrogel water uptake capacities. At 37° C.,the solfractions of the hydrogels are increased indicating an additionallost of polymer components at this incubation temperature. Thisadditional hydrogel component lost can be the consequence of the releaseof the fraction of gelatin not chemically cross-linked, since in theseconditions, the temperature (37° C.) of the hydrogel immersionincubations is above the gelatin melting point. The presence of dextransulphate in the hydrogel does not interfere with this process.

Example 12 In Vivo Biocompatibility of Cross-linked Gelatin Hydrogelsand Dextran Sulphate Containing Cross-linked Gelatin Hydrogels DuringWound Healing in Pig

GDP Film Production

In order to obtain dextran dialdehyde cross-linked gelatin hydrogel(GDP) and dextran sulphate-containing GDP films with more appropriatemechanical properties, the films are produced about 3 weeks before thestart of the experiment. One mm thick GDP films were prepared between 2glass plates by mixing gelatine (10%, final concentration) and dextrandialdehydes (5%, final concentration), both prepared in PBS and warmedat 40° C. After about one hour at room temperature, the GDP films werestored for about three weeks at 4° C. Two hours before use, the filmswere rewarmed at room temperature. Dextran sulphate-containing GDP filmsare prepared similarly to GDP films, except that dextran sulphate (MW400000-600000) is added to the dextran dialdehyde solution before mixingwith gelatin (SIPN) in order to obtain a final concentration of dextransulphate of 0.5%.

Gelatin: N°4, 4488AF2 144, from Sanofi, irradiated 0.6 Mrad in May 1995,endotoxin contents: 1.405 EU/g, 10%, final concentration, with thefollowing characteristics: 254 bloom pH 4.97, PI 7.28, obtained frombovine acid-cured tissue, after a second acid extraction (4-5 h at 55°C.), the first extraction is performed at 50° C. after a pre-treatmentat 40° C. and at pH 1-2 for 24 hours.

Dextranox: (20% oxidation, 5% final concentration, filtrated on 0.22 μm,endotoxin contents: 0.720 EU/g with a clinical grade dextran from ICNBiomedicals Inc Aurora, Ohio (MW 60000-90000, lot N° 59170), endotoxincontents: 1.700 EU/g.

Dextran sodium sulphate (MW 400000-600000), from ICN Biomedicals IncAurora, Ohio, lot N° 64914.

The Porcine Model

In this example a pig model is conducted in order to furthercharacterize and confirm the biocompatibility of dextran dialdehydecross-linked gelatin hydrogel (GDP) dressing when placed in afull-thickness wound environment. The biocompatibility of GDP isevaluated, in comparison with two largely used dressings, thehydrocolloid dressing DuoDERM and the occlusive dressing Tegaderm, bycharacterization of the intensity and/or time duration of theinflammatory reaction during wound healing. A pilot study is alsoperformed on 4 wounds to evaluate the biocompatibility of 0.5% dextransulphate-containing GDP (affinity matrix).

A porcine model is chosen because of the morphological and functionalsimilarities between pig and human skin. An unsutured full-thicknesscutaneous wound model (2 cm×2 cm square wounds) is chosen because it iseasily reproducible, and provides sufficient wound tissue to quantifyconnective tissue deposition (e.g. collagen) and cellular changes duringwound healing. Moreover, with full-thickness excisional wounds, one canmake reasonable observations on wound resurfacing by contraction andre-epithelialization.

Castrated male Belgian Landrace pig, approximately 4 months old at thetime of wounding, weighing 50 kg, are housed in controlled temperature(20° C.) and fed a maintenance diet for sows ad libitum. The pigreceives 8 cc penicillin/streptomycin as presurgery treatment. Beforesurgery, the pig receives stresnil (1 ml/20 kg), and the anaesthesia isinduced by the inhalation of halothane (4%) administered together withoxygen and nitrous oxide both at 2 l/min. After endotracheal intubation,the pig is maintained anaesthetized by the inhalation of halothane(1-2%) administered together with oxygen and nitrous oxide both at 2l/min. Premature recovery from anaesthesia is controlled by intravenousinjection of Diprivan (Propofol) until appropriate anaesthesia isobtained.

The skin of the back and sides is surgically prepared by shaving,washing with Neo-Sabimol and water, and disinfecting with 70% ethanolcontaining Hibithane. To reduce anatomical variation in the woundhealing response, 24 full-thickness square wounds (2 cm×2 cm) are made 5cm apart in the area located between the shoulder and thigh withinminimum 2.5 cm and maximum 12 cm from the pig backbone. The skin isexcised to the fascia with a scalpel, and care is taken to avoid muscleexcision.

Immediately after surgery, 4 wounds are untreated (controls), 8 woundsare treated with gelatine-dextranox polymer (3.5 cm×3.5 cm, placed overthe wounds), 4 wounds are treated with gelatine-dextranox polymer (3.5cm×3.5 cm, placed over the wounds) containing 0.5% dextran sulphate (MW:400000-600000), and 8 wounds are treated with extra thin DuoDERM (5 cm×5cm), a hydrocolloid dressing from Convatec. All the wounds are coveredwith Tegaderm, an occlusive dressing which provides for a moistenvironment, and which is obtained from 3M Medical products. Dressingsare fixed with Fixomull stretch, from Beiersdorf, and Velpo bandages toprevent possible self-trauma to the wounds.

Evaluation of Wound Healing by Planimetry and Histology

Photographs of the wounds are taken and the edges of the wounds aretraced on cellophane sheets to measure areas and perimeters of thewounds during wound healing. A public domain image processing andanalysis program (NIH Image) for Macintosh computer is used formeasurements of areas and perimeters of the wounds. This allows toevaluate quantitatively the wound resurfacing by measuring, from thedigitized contours, the change in wound area for a given period of timedue to either contraction or to both contraction andre-epithelialization, and calculate the average length of advance of thewound margin per day.

The rate of wound closure is evaluated by measuring the decrease of openwound area from the wounding time to the time of wound closure. The openwound area is given in percent of the initial wound area.

    (Open wound area (cm.sup.2) on day x/original wound area (cm.sup.2) on day 0)×100

Wound contraction is a normal process whereby the wound margins arepulled to the centre of the wound. In our porcine model, the initialsquare shape of the wound is progressively modified by contraction. Theedges of the initial square becomes concave. Edge movement is lesspronounced in the dorso-ventral direction than in the head-tailed axis.The contribution of contraction to wound closure is evaluated bymeasuring the decrease in the area (expressed as percentage of theinitial wound area) given by the sum of re-epithelialized area togetherwith the open wound area.

    [(open wound area (cm.sup.2) on day x+re-epithelialized area on day x)/original wound area (cm.sup.2) on day 0]×100

The radial progression (d) of the wound margin towards wound closure isgiven in cm/day.

    d=[(A.sub.1 -A.sub.2)/(P.sub.1 +P.sub.2)]/T

Where:

A₁ is the area of the wound at a given T₁ time after wounding

A₂ is the area of the wound at a given T₂ time after wounding

P₁ is the perimeter of the wound at T₁ time after wounding

P₂ is the perimeter of the wound at T₂ time after wounding

T is the time given in days between the two wound healing evaluations.Since the dressings are not changed during this evaluation, A₁ and P₁are the area and the perimeter of the wound at day 0, respectively.

In order to perform the histological evaluation of wound healing and theevaluation of the inflammatory response to the implant, full-thicknessskin biopsies are harvested, under general anaesthesia, at days 5, 10,15 and 23 after surgery. Ellipses are excised to include the surroundingintact skin and the whole wound. The biopsies are fixed in 4%paraformaldehyde for classical histological evaluation and picro-Siriusred staining (evaluation of the collagen fiber deposition and maturationwith the help of polarized light microscopy). The deep and large woundsproduced by biopsy removal are partially sutured and covered withTegaderm.

For classical histological evaluation, the paraformaldehyde fixedsections are stained either with hematoxylin and eosin, trichrome stain,periodic acid Schiff (PAS) stain, or Perls' Prussian blue reaction.

Body Weight and Animal Behaviour

The body weight of the pig is 50 kg the day of wounding. The body weightis not modified day 5 after surgery, but increases to 60 and 65 kg, day10 and 15 after surgery, respectively, indicating that, although 6 largebiopsies are harvested at each time of wound healing evaluation (days 5and 10 after wounding), the pig is not too much affected by the 3 firstrepetitive anaesthesia and surgical procedures. Day 23 after surgery,the body weight still remains that of day 15, indicating that finallythe harvesting of a total 18 large biopsies might stop the increase inbody weight of the animal. The days after each surgical procedure, thebehaviour of the pig had grossly returned to normal (normal activity,eating, drinking, etc).

Kinetics of Wound Closure

The days of biopsy removals (days 5, 10, 15 and 23), the rates of woundclosure, due to both contraction and re-epithelialization, arequantitatively evaluated by planimetric measurements of the remainingopen wound areas. Declining areas of the wounds plotted against the timepast wounding are shown in FIG. 15a. The kinetics of wound closure werefound to be similar for GDP, Duoderm and Tegaderm treatments, indicatingthat GDP treatment is at least as good as two of the best and largelyused dressings. Wounds treated with 0.5% dextran sulphate-containing GDPalways appear in a more advanced stage of healing. For dextransulphate-containing GDP-treated wounds as well as for Tegaderm-treatedwounds, only one wound per treatment is evaluated, at each time ofbiopsy removals. With all treatments, the wounds are closed at day 23after surgery. At this time, contraction (movement of the intact skin atthe wound edges towards the center of the wounds) accounts for about 40%of wound closure for GDP and Duoderm treated wounds, and for about 60%for dextran sulphate-containing GDP and Tegaderm treated wounds (FIG.15b). As can be seen in FIG. 16a, the reduction of the perimeters bycontraction is lower than the reduction of wound areas (FIG. 15b). Byusing the open wound areas values from FIG. 15a and the open woundperimeters of FIG. 16b, we calculate the length of daily advance of thewound margins (contraction+re-epithelialization) towards the woundcenter. As can be seen in FIG. 17, the radial progression towards woundclosure is similar for three treatments (GDP, Tegaderm and Duoderm)indicating again that GDP is a dressing at least as good as Tegaderm orDuoderm, and certainly does not interfere negatively with wound healing.The radial progression of the wounds treated with dextransulphate-containing GDP is always higher than those of GDP-, Tegaderm-and Duoderm-treated wounds, indicating that dextran sulphate-containingGDP is a good candidate to accelerate wound healing.

Wound Healing

According to our macroscopic evaluations, GDP does not interferenegatively with wound healing in pig, the healing of GDP-treated woundsis comparable to the healing of wounds treated with two good dressings(Duoderm and Tegaderm). The healing of the wounds treated with dextransulphate-containing GDP is always in a more advanced stage than thehealing of GDP, Tegaderm and Duoderm-treated wounds, indicating thatdextran sulphate-containing GDP is a good candidate to accelerate woundhealing.

Histological evaluations of wound biopsies show that inflammatory cells,inflammatory foci and granulomas are always, and by far, more abundantin the granulation and the cellular scar tissues of Duoderm-treatedwounds. Both cavities containing particles of Duoderm material andgranulomas with numerous foreign-body giant cells are characteristic forthe scar tissue of Duoderm-treated wounds. Inflammatory foci are alsopresent in the granulation and the scar tissues of dextransulphate-containing GDP-, GDP- and Tegaderm-treated wounds, but they areless abundant than in Duoderm-treated wounds and contain less foamymacrophages and foreign-body giant cells. Abundance of inflammatory fociis: Duoderm>GDP, dextran sulphate GDP, Tegaderm. In conclusion, a weakto moderate foreign body reaction is observed with Dextransulfate-containing GDP- and GDP-treated wounds. By contrast, a strongforeign body reaction is seen in the Duoderm-treated wounds. GDP anddextran sulphate-containing GDP can thus be considered as biocompatibledressings. In this example, dextran sulphate containing GDP-treatedwounds re-epithelialize faster than DuoDERM and Tegaderm treated woundsindicating that dextran sulphate-containing GDP is a better candidate toaccelerate wound healing. Moreover, on the basis of the collagenorganisation in the scar tissue and the presence of a basement membraneunder the new epithelium, dextran sulfate-containing GDP-treated woundsare always found to be in a more advanced stage of wound haling.

What is claimed is:
 1. A medicament containing a biopolymer matrixcomprising gelatin cross-linked with an oxidized polysaccharide andwherein a polysulfated polysaccharide with a MW greater than 30,000 kDais mechanically entrapped during the formation of said matrix, therebyforming a semi-interpenetrating polymer network.
 2. A medicamentcontaining a biopolymer matrix of claim 1 wherein said oxidizedpolysaccharide is an oxidized dextran or an oxidized xanthan.
 3. Amedicament containing a biopolymer matrix of claim 1 wherein additionalcompounds are attached to and/or incorporated into said matrix, withsaid additional compounds being selected from the group consistingabiocompatible polyanion which has the capacity to bind heparin-bindinggrowth factors; a proteoglycan containing glycosaminoglycan chainscapable of binding to heparin-binding growth factors; a functionalanalog of heparin which can bind or stabilize heparin-binding growthfactors and a monoclonal or polyclonal antibody or a microproteinobtainable by phage display that have a high and selective affinity formolecular factors that can modulate the wound healing process.
 4. Amedicament containing a biopolymer of claim 1 wherein said matrix is inthe form of a hydrated film.
 5. A medicament containing a biopolymer ofclaim 1 wherein said matrix is in the form of a hydrated or dry foam. 6.A medicament containing a biopolymer of claim 1 wherein said matrix isin the form of dry fibers, which may be fabricated into a woven ornon-woven tissue.
 7. A medicament containing a biopolymer matrix ofclaim 1 wherein said matrix is in the form of hydrated or dry microbeads.
 8. A medicament containing a biopolymer matrix of claim 1 whereinsaid matrix is in the form of a dry powder.
 9. A medicament containing abiopolymer matrix of claim 1 into which a therapeutically effectiveamount of a drug is non-covalently incorporated.
 10. A medicamentcontaining a biopolymer matrix of claim 1 into which a therapeuticallyeffective amount of a wound healing-stimulating drug is incorporated.11. A medicament containing a biopolymer matrix of claim 3 wherein atleast one of the additional compounds is selected from the groupconsisting of EGF-like factors, FGF-like factors, TGF-β-factors,IGF-like factors, PDGF-like factors, VEGF-like factors, keratinocytecell lysate and purified keratinocyte lysate.
 12. A medicamentcontaining a biopolymer matrix of claim 3 wherein at least one of thecompounds is an antiseptic.
 13. A medicament containing a biopolymer ofclaim 1 in the form of a wound dressing and/or controlled releasedevice.
 14. A medicament of claim 13 in the form of a controlled or slowrelease for transdermal drug delivery.
 15. A controlled or slow releasedevice of claim 14 comprising micro particles loaded with a drug whichcan be injected intravenously, subcutaneously or intramuscularly.
 16. Acontrolled or slow release device of claim 14 comprising micro particlesloaded with a vaccine which can be injected intravenously,subcutaneously or intramuscularly.
 17. A method of treating skin woundsor dermatological disorders of warm-blooded animals comprising applyingto skin wounds on the skin of warm-blooded animals a medicament of claim10.